Congenital Heart Disease (CHD) is the most common class of life-threatening birth defect. Whereas hundreds of genes have been implicated in CHD, the mechanistic basis of abnormal cardiac morphogenesis causing CHD is unknown in almost all cases. Our work on Hedgehog signaling and CHD over the last decade has culminated in the novel hypothesis that loss of Hedgehog signaling causes a failure of stereotypical control of cardiac progenitor differentiation timing as an underlying cause of CHD. This work may highlight molecular control of differentiation timing as a cornerstone of cardiac development with defects in differentiation timing as a candidate mechanism underlying CHD etiology. This proposal is formed from a decade of study of the molecular mechanisms underlying Atrioventricular septal defects (AVSDs). AVSDs are a serious form of CHD in humans, comprising 5-10% of all CHD and a greater proportion of cases with significant morbidity and mortality1. We have previously contributed to a paradigm shift in the understanding of AV septation, demonstrating that cilia-based Hedgehog (Hh) signaling is required in second heart field (SHF) cardiac progenitors, rather than in the heart itself, for AV septation. Our laboratory has implicated cilia, Hedgehog signaling, and cardiogenic transcription factors in the SHF for AV septation. In preliminary results, our recent work demonstrates that Hh signaling controls SHF progenitor differentiation delay and that removal of Hh signaling causes precocious cardiac differentiation. In this proposal we harness this novel paradigm for cardiac differentiation control to address the genetic and molecular mechanisms underlying cardiac morphogenesis. In Specific Aim 1, we directly interrogate the relationship between cardiac differentiation and cardiac morphogenesis to determine the relationship between Hh signaling, cardiac progenitor differentiation control, and cardiac morphogenesis. In Specific Aim 2, we will investigate the Forkhead box transcription factor gene Foxf1 as a Hh-target gene and candidate mediator of SHF differentiation delay; and in Specific Aim 3, we will identify the diversity of developmental lineages in the SHF and in which lineages Hh signaling acts as a differentiation control switch. If successful, these aims will contribute to a mechanistic understanding of AVSDs and support a novel paradigm for Hh signaling control of differentiation timing. !